The advent of DNA sequencing technology has revolutionized molecular biology through vastly extending the capability to identify and delineate DNA compositions from any biological sources. Nowadays, DNA sequencing is routinely employed in the course of conducting scientific research, and it is becoming more commonly practiced in clinical diagnostics, environmental studies, and forensic investigations.
Sanger's dideoxy termination method (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74: 563-5467 (1977)) and Maxam-Gilbert's chemical degradation method (Maxam and Gilbert, Proc. Natl. Acad. Sci. U.S.A. 74: 560-564 (1977)) are the two traditional procedures commonly practiced in the field. Both methods require four samples with each sample containing a family of DNA strands in which all strands terminate in the same nucleotide. Upon termination of the polymerization reactions, gel electrophoresis, or more recently capillary array electrophoresis is used to resolve the different length strands terminated at different base positions and to determine the nucleotide sequence, either by differentially tagging the strands of each sample before electrophoresis to indicate the terminal nucleotide, or by running the samples in different lanes of the gel or in different capillaries. These procedures are labor- and time-intensive. Improved DNA sequencing techniques that overcome the limitations of electrophoresis have been developed. Such techniques include pyrosequencing, MS sequencing (Fu, D. J. et al. (1998) National Biotechnol. 16, 381-384; Roskey, M. T. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4724-4729; J. R., Itagaki et al. (2001) Nucleic Acids Res. 29, e104), sequencing by hybridization (Drmanac, S. et al. (1998) Nat. Biotechnol. 16, 54-5810), sequence-specific detection of single-stranded DNA using engineered nanopores.
More recently, new DNA sequencing approaches based on a solid surface have been reported. One example of this approach involves the use of nucleotides or nucleotide analogues known as chain or extension terminators. These extension terminators prevent further addition by the polymerase of nucleotides or nucleotide analogs to the 3′ end of the nascent DNA strand once they are incorporated into the nascent strand. In practice, in order to obtain consecutive base sequences of a template DNA, the extension terminators must be washed off before the next round of base incorporation event can take place. See, for example, U.S. Pat. Nos. 5,302,509 and 6,087,095. The requisite washing step between each base incorporation event is thus the rate-limiting step, which inevitably delays the sequencing process. Furthermore, the washing step causes the displacement of costly reagents in the reaction including polymerases and nucleotide analogs.
Thus, there remains a considerable need for alternative methods and devices designed to perform high-throughput and cost-effective nucleic acid sequencing.